Automated clinical analyzers relying upon radio-metric detection of a change in absorption require calibration using a "standard" or calibrator. Such calibrators usually require two or more known concentrations of an analyte of choice, such as glucose, to be fixed at specific levels of the measured characteristic, e.g., reflection or transmission density. From these a "calibration" curve is derived for use with readings for unknown analyte concentrations.
It is conventional, in the preparation of calibrators, to separate the small molecular components and ions from the blood serum matrix and then to add back to the matrix the materials of choice to certain pre-selected levels for that particular calibrator. Such separation commonly is achieved by dialysis. "Dialysis" as used in this application includes not only non-pressurized equalibrating flow of such small molecular components through a semi-permeable membrane, filter or permeable gel away from the matrix, but also ultrafiltration and diafiltration which rely upon a pressure assist to force such small molecular components to flow through the filter away from the matrix. Dialysis separation has now been found to strip materials which complex or otherwise bind with various components of the matrix, for example, bilirubin which complexes with matrix protein, e.g., albumin. Such stripping leaves the protein of the matrix with vacant sites capable of complexing or binding a variety of other organic molecules. The binding capability of these sites was known, however, only with respect to certain specific materials.
It is these dialyzed serum based calibrators which have been found to create a problem when used with various kinds of analytical reagents, whether the reagents are in liquid form in a container or are disposed in dried form in a test element. The reagents are relied upon to create a radiometrically-detectable change, and accordingly must be readable by a radiometer at a known wavelength of detection. The presence in a calibrator of a protein with free binding sites can remove the reagent from the detection area, particularly when using the dried, test element form. The reagent can also shift its wavelength of absorption away from the expected wavelength by binding to the free sites. A wavelength shift is a problem no matter whether the reagents are in liquid form or dried prior to use.
Considering first the nondetection of the reagents, this occurs, for example, in certain dried test elements used for assaying analytes, wherein an opaque porous overlayer is applied to a reagent layer. The porosity of the opaque layer may be such that the protein of a serum-based calibrator, when applied thereto, is retained. When this happens, free sites on the retained protein, e.g., retained albumin, can attract and bind the indicator reagent if such reagent is migratable. The porous layer retaining the albumin in effect competes with the reagent layer for the reagent used for detection. Less reagent is available for detection in the reagent layer, and it cannot be detected in the opaque overlayer.
Examples of dry test elements susceptible to the phenomenon of applied albumin being retained in an opaque overlayer where it attracts indicator dye, are disclosed in U.S. Pat. No. 3,992,158 issued Nov. 16, 1976. This patent describes a multilayer analytical element comprising in one embodiment thereof an opaque spreading layer and a reagent layer, the element being capable of producing a dye density which is proportional to the concentration of the analyte of choice when the spreading layer is contacted with a drop of sample solution. Such an element can be used to detect quantitatively a wide variety of analytes, through the use of, for example, migratable dyes. Such dyes migrate between the spreading layer and the reagent layer in the presence of liquid. The problem is that when calibrators comprising a dialyzed serum matrix or albumin are used with these analytical elements, a consistent negative bias occurs. That is, the amount of dye which is detected is always less than the amount one would expect. Such negative bias in many cases falls outside acceptable ranges of accuracy. This effect occurs wherever a migratable dye is used, so that it is not limited necessarily to the assay for any one particular analyte, such as glucose.
Other analytical elements capable of producing the same negative bias when calibrated with a dialyzed serum matrix include, for example, those shown in U.S. Pat. No. 3,802,842, issued Apr. 9, 1974, and Belgian Patent No. 837,939, published on May 14, 1976.
As noted, a second undesirable aspect of the binding of dyes to the free sites of the protein is that such binding tends to cause a shift in absorption wavelengths. If the analysis is based upon the amount of absorbance occuring at the wavelength before the shift, any binding of the substance will reduce the amount of absorbance that is detected. Bilirubin is one example. Unbound bilirubin has its maximum absorbance at 435 nm, while bilirubin bound to ablumin has such absorbance at 460 nm.
Various organic materials such as bilirubin, phenolsulfophthalein dyes, 1-anilino-8-naphthalene sulfonate, fatty acids and L-thyroxine which are known to bind to the active sites of an albumin molecule are discussed in Kragh-Hansen et al "Protein Binding of Small Molecules", Biochimica et Biophysica Acta. 365 (1974) p. 360.